Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in a range of biotechnological fields. A particularly important area is the generation of nucleotide mutants and the screening for and identification of such mutants. This in turn has implications, for example, in understanding the genetic basis behind certain disease conditions which is becoming of increasing relevance as the human genome is progressively sequenced.
An efficient and accurate method of mutation detection is crucial in implicating disease candidate genes and in the screening programs which follow identification of disease causing mutations. Many human inherited and sporadic disorders are caused by small mutations including base substitutions, additions and deletions. Among these disorders are the Mendelian single gene disorders, sporadic somatic mutations causing cancers and complex genetic traits. Whilst some diseases are caused by a limited and well characterised set of mutations, most genetic diseases are caused by one or more of a large range of mutations occurring anywhere within the gene. It is important, therefore, that a mutation detection protocol be able to scan a region of DNA, identify any change and describe the resulting nucleotide differences from wild-type. With the increasing use of population molecular genetics and as clinicians begin to use mutation analysis as a clinical tool, there is a need to develop mutation detection protocols which can be automated, are less dependant on user expertise and are more accurate and reliable.
Current mutation detection protocols require either a gel based detection system or sequence specific primers. Gel based detection methods include direct sequencing of amplified DNA fragments and various techniques involving either cleavage of mismatched bases in heteroduplexes or mobility differences of single or partially denatured DNA strands.
Detection of mutations by DNA sequencing can provide good results in relation to accuracy and information about the position and nature of the mutation (Hattori et al, 1993), however, although advances have been made in this area, the technique is not fully automated and is labour intensive. Most mutations occur as heterozygotes and there are technical difficulties with the ability of currently available computer software to identify two different nucleotide bases at a mutated residue.
Many mutation detection techniques exploit differential electrophoretic mobilities of DNA fragments with sequence differences. Single strand conformation polymorphism (SSCP) exploits the fact that the secondary structure of a single strand of DNA is sequence based and, therefore, strands with even just one base difference will migrate at a different rate (Orita et al, 1989). This technique is again gel based and can lack sensitivity. Furthermore, the method cannot be readily automated and requires a large amount of labour due to the necessary gel step which in most cases must be optimised to the specific sample being analysed. They also do not give any information about the position or nature of the change and do not routinely identify all mutations.
Mutation detection based on the identification of base pair mismatches in heteroduplex DNA strands is another method of identifying point changes. There are a number of techniques available that cleave DNA at mismatched base pairs in heteroduplex DNA. Mismatch cleavage protocols include chemical and enzymatic mismatch cleavage. The techniques are also gel based. The chemical cleavage method uses osmium tetroxide to cleave at the mismatched base (Cotton et al, 1988) followed by separation of cleaved products on denaturing gels. A major disadvantage of the chemical cleavage protocol is the use of extremely toxic chemicals.
Other methods for detection of known mutations include minisequencing allele specific polymerase chain reaction (PCR), oligonucleotide probe arrays (Lipshutz et al, 1995) which requires knowledge of the sequence of wild-type and mutant. Although this technique is suitable for non-gel based detection methods, it is only useful for know mutations. Furthermore, the large number of oligonucleotides required to cover all known mutations in many genes makes this approach prohibitively expensive and labour intensive.
With the development of the matrix assisted laser desorption ionisation—time of flight mass spectrometer (MALDI-TOF MS), the ability to accurately determine the mass of biomolecules of a limited size has been achieved. Although detection of DNA fragments of up to 622 base pairs in length has been reported, large fragments cannot be accurately sized and a mass accuracy of ±3 bp is quoted (Liu et al, 1995). This level of accuracy is clearly insufficient for the detection and characterisation of base substitutions.
There is a need, therefore, to develop an effective and accurate means of detecting mutations in nucleic acid molecules. Preferably, the mutation detection system would be automatable.
In work leading up to the present invention the inventors developed a mutation detection system which exploits the accuracy of mass determination of MALDI-TOF MS and which is applicable for large DNA fragments. The method of the present invention do not require gel electrophoresis nor is prior knowledge of the nucleotide sequence necessary. The method of the present invention is also capable of being automated.